Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial but also community acquired pathogen that can cause serious infections such as surgical wound infections, pneumonia, endocarditis and septicemia. Resistance to methicillin is due to the presence of the mecA gene that encodes a modified Penicillin-Binding protein, PBP2a or PBP2′, with reduced affinity for B-lactam drugs. The mecA gene is carried by a cassette named the SCCmec (Staphylococcal Cassette Chromosome mec; Ito et al., 2001, Antimicrob. Agents Chemother. 45(5):1323-1336; Hiramatsu, et al., 2001, Trends Microbiol. October; 9(10):486-93), a mobile element that can be incorporated into the chromosome of S. aureus and other coagulase negative Staphylococci, mainly S. epidermidis and S. haemolyticus. SCCmec is characterized by the presence of terminal inverted and direct repeats, a set of site-specific recombinase genes (ccrA and ccrB), and the mecA gene complex (Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458; Katayama et al., 2000, Antimicrob. Agents Chemother. 44:1549-1555). The site of insertion of this mecA gene cassette SCCmec into the Staphylococcus aureus genome is known and the sequence conserved (Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336). After insertion into the S. aureus chromosome, the SCCmec has a left extremity junction region and a right extremity junction region (see FIG. 1), where the SCCmec sequence is contiguous with the S. aureus chromosomal sequence. The nucleotide sequence of the regions surrounding the left and right boundaries of SCCmec DNA (i.e. attL and attR, respectively), as well as those of the regions around the SCCmec DNA integration site (i.e., attBscc, the bacterial chromosome attachment site for SCCmec DNA), have previously been analyzed. Sequence analysis of the integration sites revealed that attBscc is located at the 3′ end of a novel open reading frame (ORF), orfX. orfX encodes a putative 159-amino acid polypeptide that exhibits sequence homology with some previously identified polypeptides of unknown function (Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458). Organization of the mecA region of SCCmec has additionally been studied (Oliveira, D. C., et al., 2000, Antimicrob. Agents Chemother. 44(7):1906-1910).
MRSA can be carried by healthy people without causing any disease but these healthy carriers, when entering the hospital, can contaminate hospitalized patients. Additionally, a patient can contaminate himself, e.g., if one undergoes surgery, the risk of infection is increased. MRSA healthy carriers constitute a reservoir of MRSA and screening of these carriers must be performed to eradicate the strains by local decontamination. MRSA screening is now recognized as a major tool to reduce the prevalence of MRSA strains in the world. Typically, in an MRSA assay in a patient, a nasal swab is taken from the patient and cultured repeatedly, to determine if an MRSA strain is present. The need to culture could be obviated by an assay for identifying MRSA directly from a nasal swab. Culture identification methods typically require minimally 24 hours, and more typically 72 hours, to obtain results. New chromogenic media (having substrate(s) within the media and, typically, antibiotic (e.g., cefoxitin) to select methicillin-resistant strains) can potentially restrict this time to result to a 24-48 hour time period. However, in the case of MRSA infection, results are needed in a matter of hours, since the patient should be isolated until results are obtained. Therefore, a reliable molecular MRSA test which can provide results in a matter of 2-4 hours is highly desirable.
Amplification is a well known art, and various methods have been developed, including transcription-based amplification such as transcription-mediated amplification (TMA; U.S. Pat. Nos. 5,766,849 5,399,491; 5,480,784; 5,766,849; and 5,654,142) and nucleic acid sequence-based amplification (NASBA; 5,130,238; 5,409,818; 5,654,142; and 6,312,928), and cycling nucleic acid amplification technologies (thermocycling) such as polymerase chain reaction (PCR; U.S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202) and ligase chain reaction (LCR; U.S. Pat. No. 5,792,607). Known amplification methods also include strand displacement amplification (SDA), self-sustained sequence replication (3SR), Q-β replicase, and cascade rolling circle amplification (CRCA).
Detection methods utilizing nucleic acids are also well known in the art. Nucleic acids are often labeled for various detection purposes. For example, methods described in U.S. Pat. No. 4,486,539 (Kourlisky); U.S. Pat. No. 4,411,955 (Ward); U.S. Pat. No. 4,882,269 (Schneider) and U.S. Pat. No. 4,213,893 (Carrico), illustrate preparation of labeled detection probes for detecting specific nucleic acid sequences. Probe designs for different detection methods, such as target-capture, HPA, TAQman, molecular beacons and sandwich hybridization have also been described (e.g., U.S. Pat. No. 4,486,539, and U.S. Pat. Nos. 4,751,177; 5,210,015; 5,487,972; 5,804,375; 5,994,076). Nucleic acid hybridization techniques and conditions are known to the skilled artisan and have been described for example, in Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Lab. Press, December 1989; U.S. Pat. No. 4,563,419 (Ranki) and U.S. Pat. No. 4,851,330 (Kohne) and in Dunn, et al., Cell 12, pp. 23-26 (1978) among many other publications. Probe designs for different detection methods are also known, such as target-capture, HPA, TaqMan, molecular beacons and sandwich hybridization (e.g., U.S. Pat. No. 4,486,539, and U.S. Pat. Nos. 4,751,177; 5,210,015; 5,487,972; 5,804,375; 5,994,076).
Earlier molecular methods developed to detect and identify MRSA based on the detection of the mecA gene and S. aureus-specific chromosomal sequences have been described. (Saito et al., 1995, J. Clin. Microbiol. 33:2498-2500; Ubukata et al., 1992, J. Clin. Microbial. 30:1728-1733; Murakami et al., 1991, J. Clin. Microbiol, 29:2240-2244; Hiramatsu et al., 1992, Microbial. Immunol. 36:445-453). However, positive results for the presence in a sample of both mecA gene and S. aureus chromosomal sequences cannot guarantee MRSA is present, since, for example, in tests based on the detection of mecA and S. aureus specific marker, false positives can be observed in the presence of MSSA and methicillin resistant coagulase negative Staphylococcus that possess the mecA gene. Furthermore, in tests based on the detection of the cassette junction only, false positives have been observed with methicillin-susceptible S. aureus isolates containing a small fragment of the right extremity of the SCCmec (see Rupp, J. et al., J. Clin. Microbiol. 44(6): 2317 (2006)). Additionally, Ramakrishnan and Riccelli describe a method for detecting MRSA utilizing oligonucleotide probes having sequences that are complementary to regions near the left junction of the SCCmec cassette insertion site, including part of the SCCmec cassette sequence and part of the S. aureus sequence in the region of insertion (the left extremity junction region) (U.S. patent publication No. US20060057613).
However, previous attempts to determine MRSA by molecular methods have had difficulties with false positive results. Such results have been postulated to be the result of any of the presence of a mixed population in swabs, the presence in an MSSA of a residual SCCmec right extremity fragment following the deletion of the mecA gene and/or non-specific amplification. To date, two concepts for determining resistance to methicillin carried specifically by S. aureus have been published:
the SCCmec right extremity junction amplification concept (Hiramatsu et al. WO97/31125; EP 0 887 424; U.S. Pat. No. 6,156,507; and further, Huletsky and Rossbach WO02/099034 (2002); Huletsky et al. J. Clin. Microbiol. 42(5): 1875-1884 (2004))
the immuno-enrichment concept described by François and co-workers (François, P et al. J. Clin. Microbiol. 41(1):254-260 (2003); WO02/082086), in which the immuno-enrichment is followed by amplification of three markers (mecA gene, S. aureus-specific marker, and S. epidermidis-specific marker).
The SCCmec right extremity junction concept is based on the amplification of a region covering the right extremity junction region of the SCCmec integration site. The principle is the following: the SCCmec cassette always integrates the S. aureus chromosome upstream of a S. aureus specific open reading frame called orfX; the PCR assay combines multiple forward primers located on the right part of the cassette, one reverse primer and a beacon probe, both located in the S. aureus chromosomal orfX, i.e., downstream of the right extremity junction of SCCmec with orfX (“right extremity junction region” of orfX). Hiramatsu et al, describe a test with two forward primers in the right extremity junction region of the cassette to amplify the main SCCmec types described at that time (one primer for SCCmec types I and II and a second primer for type III). Huletsky et al. set forth that several MRSA strains were not detected if only the two forward primers described by Hiramatsu were used, and they determined new types of cassettes named as MREJ types having sequence variations in the right part of the SCCmec cassette. A commercially available (Infectio Diagnostics Inc.) test combines (refer to FIG. 1) five forward primers located in the right part of the cassette (one primer was designed for the detection of MREJ types i and ii and the four others for the MREJ types iii, iv, v and vii), one reverse primer located in the orfX, and three generic beacons covering the same portion of the orfX region and required to identify the orfX variants identified. This test is performed in real-time PCR. However, the specificity of this test as reported (Huletsky et al. 2004) shows that 4.6% of MSSA (26 out of 569 tested) were misidentified. False-positive result has also been reported with another commercial test using a single-locus (right extremity SCCmec cassette-orfX junction) PCR assay (Rupp, J, et al., J. Clin. Microbiol. (44)6: 2317 (2006)).
Thus false positives remain an issue, and there is a strong need for an improved test for MRSA to reduce false positive results obtained with current tests. The challenge for such a test is that, due to the presence of a mixed population in nasal swabs, the following mixtures can be present: one or more of (1) MRSA, (2) methicillin sensitive coagulase-negative Staphylococci (e.g., methicillin sensitive Staphylococcus epidermidis (MSSE)), (3) methicillin sensitive Staphylococcus aureus (MSSA), and (4) methicillin resistant coagulase-negative Staphylococci (MR-CNS) (mainly methicillin resistant Staphylococcus epidermidis (MRSE)). Furthermore, there has been report of clinical MSSA isolates retaining SCCmec elements without the mecA gene. (Donnio, P.-Y., et al., J Clin Microbiol. 2005 August; 43(8): 4191-4193). Because only the presence of a MRSA will lead to decontamination of the carrier, the test must ensure that resistance to methicillin is carried by S. aureus and not by S. epidermidis (or coagulase (−) Staphylococcus strain). Thus, also, amplification and detection of the mecA gene (associated or not with a S. aureus specific marker) directly on the mixed population present on the swabs is not appropriate; indeed in both situations (MRSA+MSSE or MSSA+MRSE), both markers (mecA and S. aureus specific marker) will be detected whereas only the first situation with an MRSA is desired to be specifically detected by the clinician. The present invention addresses primary sources of MRSA false positives and thus provides a much-needed, improved test to detect MRSA that has not been addressed by currently available tests.